Treponema pallidum subspecies pallidum (T. pallidum), the causative agent of syphilis, is one of the few major bacterial pathogens that has not been cultured continuously in vitro. Currently, T. pallidum must be propagated by inoculation of rabbits for use in research studies or for diagnostic or epidemiologic purposes. The lack of continuous in vitro culture severely restricts studies of the organism?s biology and pathogenic mechanisms and precludes application of many valuable technologies, including mutational analysis. A system involving co-culture of T. pallidum with Sf1Ep rabbit epithelial cells under microaerobic conditions promotes survival and multiplication of T. pallidum for up to 18 days. However, repeated attempts to expand the yield or longevity of growth (using refinement of growth conditions, subculture, medium exchange, or other tactics) have not been successful. In this project, novel approaches will be utilized to further define the growth characteristics and requirements and gene expression patterns of T. pallidum. In the first Aim, recently developed methods for computational analysis of microscopy images and assessment of cell wall synthesis and DNA synthesis will be employed to gain a better understanding of the growth characteristics of T. pallidum in axenic and Sf1Ep cell co-culture conditions and to identify cell processes that are restricted during in vitro growth. In addition, co-incubation with tissue explants, as successfully applied to Borrelia burgdorferi studies, will be examined as a means to provide a ?tissue-like? environment that promotes T. pallidum in vitro multiplication. The second Aim will employ the recently developed differential RNA-seq (dRNA-seq) method to globally determine the transcriptional start sites in the T. pallidum genome. dRNA-seq will be applied to samples from infected rabbits as well as T. pallidum incubated in vitro in different oxygen levels, temperatures, nutrient compositions, and mammalian cell conditions. The resulting information will be invaluable in defining the poorly understood promoters and regulatory elements of this organism, including the potential identification of regulatory RNA species. In addition, comparison of transcription profiles from in vivo and in vitro grown organisms may provide fruitful directions for the in vitro growth studies of Aim 1. The research team for this project includes experts in the assessment of bacterial cell growth and division, as well as in the global analysis of gene transcription in bacteria. We anticipate that the combination of cell biology and RNA analysis approaches will provide new insights into the unique physiology of T. pallidum and will aid in attempts to culture the organism in vitro.